1. Field of the Invention
The present invention relates to subtilase enzymes having an additional amino acid in the active site loop (b) region from position 95 to 103 and detergent and cleaning compositions comprising same. The invention further relates to genes coding for the expression of said enzymes when inserted into a suitable host cell or organism; and host cells transformed therewith, and methods for producing the enzymes.
2. Description of the Related Art
In the detergent industry, enzymes have been used in washing formulations for more than 30 years. Such enzymes include proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. The most important commercially are proteases.
An increasing number of commercially used proteases are protein engineered variants of naturally occurring wild-type proteases, e.g. DURAZYM(copyright) (Novo Nordisk A/S), RELASE(copyright) (Novo Nordisk A/S), MAXAPEM(copyright) (Gist-Brocades N.V.), PURAFECT(copyright) (Genencor International, Inc.).
In addition, a number of protease variants have been described in the art, such as in EP 130756 (GENENTECH) (corresponding to U.S. Reissue Pat. No. 34,606 (GENENCOR)); EP 214435 (HENKEL); WO 87/04461 (AMGEN); WO 87/05050 (GENEX); EP 260105 (GENENCOR); Thomas, Russell, and Fersht, Nature, 318, 375-376 (1985); Thomas, Russell, and Fersht, J. Mol. Biol., 193, 803-813 (1987); Russel and Fersht, Nature, 328, 496-500 (1987); WO 88/08028 (Genex); WO 88/08033 (Amgen); WO 95/27049 (SOLVAY S.A.); WO 95/30011 (PROCTER and GAMBLE COMPANY); WO 95/30010 (PROCTER and GAMBLE COMPANY); WO 95/29979 (PROCTER and GAMBLE COMPANY); U.S. Pat. No. 5,543,302 (SOLVAY S.A.); EP 251 446 (GENENCOR); WO 89/06279 (NOVO NORDISK A/S); WO 91/00345 (NOVO NORDISK A/S); EP 525 610 A1 (SOLVAY); and WO 94/02618 (GIST-BROCADES N.V.).
However, even though a number of useful protease variants have been described, there is still a need for new improved proteases or protease variants for a number of industrial uses.
Therefore, an object of the present invention is to provide improved proteases or protein engineered protease variants, especially for use in the detergent industry.
The present inventors have found that subtilisins wherein at least one of the active site loops is longer than those presently known, exhibit improved wash performance properties in detergent compositions. The identification thereof was done by constructing subtilisin variants, especially of subtilisin 309 (BLSAVI or SAVINASE(copyright)), which exhibited improved wash performance properties in detergent compositions relative to the parent wild-type enzyme. This was described in our earlier application DK 1332/97, which published as WO 99/27082.
It has now been found that certain subtilases or variants thereof of the I-S1 (true xe2x80x9csubtilisinsxe2x80x9d) and I-S2 (high alkaline subtilisins) sub-groups having at least one additional amino acid residue in the active site loop (b) region from position 95 to 103, exhibit surprisingly improved wash performance in comparison to those presently known and those described in said application.
The improved proteases according to the invention may be obtained by isolation from natural resources or by the introduction of at least one further amino acid residue (an insertion) in the active site loop (b) region in a wild-type subtilase (for a definition of the active site loops and the numbering of positions see below).
Although this finding was done in subtilisin 309, it is predicted that it will be possible to produce or isolate similar advantageous subtilases or subtilase variants.
Furthermore it will be possible to specifically screen natural isolates to identify wild-type subtilases comprising an active site loop (b) region which is longer than the corresponding active site loop region in known wild-type subtilases, such as subtilisin 309, which subtilases can be considered to have an inserted amino acid residue in the active site loop (b) region, and exhibiting excellent wash performance in a detergent, in comparison to their closest related known subtilisin, such as subtilisin 309.
Concerning alignment and numbering reference is made to FIGS. 1A, 1B, 2A and 2B showing alignments between subtilisin BPNxe2x80x2 (BASBPN) (a) and subtilisin 309 (BLSAVI) (b), and alignments between subtilisin BPNxe2x80x2 (a) (BASBPN) and subtilisin Carlsberg (g) (BLSCAR). These alignments are used herein as a reference for numbering the residues.
The seven active site loops (a) to (g) are herein defined as the segments of amino acid residues provided below (including the terminal amino acid residues):
(a) the region between amino acid residue 33 and 43;
(b) the region between amino acid residue 95 and 103;
(c) the region between amino acid residue 125 and 132;
(d) the region between amino acid residue 153 and 173;
(e) the region between amino acid residue 181 and 195;
(f) the region between amino acid residue 202 and 204;
(g) the region between amino acid residue 218 and 219.
Accordingly, in a first aspect the invention relates to an isolated (i.e. greater than 10% pure) subtilase enzyme of the I-S1 and I-S2 sub-groups having at least one additional amino acid residue in the active site loop (b) region from position 95 to 103, whereby said additional amino acid residue(s) corresponds to the insertion of at least one amino acid residue.
In a second aspect the invention relates to an isolated DNA sequence encoding a subtilase variant of the invention.
In a third aspect the invention relates to an expression vector comprising an isolated DNA sequence encoding a subtilase variant of the invention.
In a fourth aspect the invention relates to a microbial host cell transformed with an expression vector according to the third aspect.
In a further aspect the invention relates to the production of the subtilisin enzymes of the invention.
The enzymes of the invention can generally be produced by either cultivation of a microbial strain from which the enzyme was isolated and recovering the enzyme in substantially pure form; or by inserting an expression vector according to the third aspect of the invention into a suitable microbial host, cultivating the host to express the desired subtilase enzyme, and recovering the enzyme product.
Further the invention relates to a composition comprising a subtilase or subtilase variant of the invention.
Even further the invention relates to the use of the enzymes of the invention for a number of industrial relevant uses, in particular for use in cleaning and detergent compositions, comprising the subtilisin enzymes of the present invention.
Definitions
Prior to discussing this invention in further detail, the following terms and conventions will first be defined.
Nomenclature and Conventions for Designation of Variants
In describing the subtilases of the present invention, the following nomenclatures and conventions have been adapted for ease of reference:
A frame of reference is first defined by aligning the isolated or parent wild-type enzyme with subtilisin BPNxe2x80x2 (BASBPN).
The alignment can be obtained by the GAP routine of the GCG package version 9.1 to number the variants using the following parameters: gap creation penalty=8 and gap extension penalty=8 and all other parameters kept at their default values.
Another method is to use known recognized alignments between subtilases, such as the alignment indicated in WO 91/00345. In most cases the differences will not be of any importance.
Such alignments between subtilisin BPNxe2x80x2 (BASBPN) and subtilisin 309 (BLSAVI) and subtilisin Carlsberg (BLSCAR), respectively are indicated in FIGS. 1A, 1B, 2A, and 2B. They define a number of deletions and insertions in relation to BASBPN. In FIG. 1A, subtilisin 309 has 6 deletions in positions 36, 58, 158, 162, 163, and 164 in comparison to BASBPN, whereas in FIG. 1B subtilisin 309 has the same deletions in positions 36, 56, 159, 164, 165, and 166 in comparison to BASBPN. In FIG. 2A subtilisin Carlsberg has one deletion in position 58 in comparison to BASBPN, whereas in FIG. 2B subtilisin Carlsberg has the one deletion in position 56 in comparison to BASBPN. These deletions are indicated in FIGS. 1A, 1B, 2A, and 2B by asterisks (*).
The various modifications performed in a wild-type enzyme are indicated in general using three elements as follows:
Thus, the notation G195E means a substitution of glycine in position 195 with glutamic acid.
In the case when the original amino acid residue may be any amino acid residue, a short hand notation may at times be used indicating only the position and substituted amino acid,
Such a notation is particularly relevant in connection with modification(s) in homologous subtilases (vide infra).
Similarly when the identity of the substituting amino acid residue(s) is immaterial, the following short hand notation can be used:
When both the original amino acid(s) and substituted amino acid(s) may comprise any amino acid, then only the position is indicated, e.g., 170.
When the original amino acid(s) and/or substituted amino acid(s) may comprise more than one, but not all amino acid(s), then the selected amino acids are indicated inside brackets { },
For specific variants the specific three or one letter codes are used, including the codes Xaa and X to indicate any amino acid residue.
Substitutions:
The substitution of glutamic acid for glycine in position 195 is designated as:
Gly195Glu or G195E
or the substitution of any amino acid residue acid for glycine in position 195 is designated as:
Gly195Xaa or G195X or Gly195 or G195
The substitution of serine for any amino acid residue in position 170 would thus be designated
Xaa170Ser or X170S or 170Ser or 170S.
Thus, 170Ser comprises e.g. both a Lys170Ser modification in BASBPN and an Arg170Ser modification in BLSAVI (cf. FIG. 1).
For a modification where the original amino acid(s) and/or substituted amino acid(s) may comprise more than one, but not all amino acid(s), the substitution of glycine, alanine, serine or threonine for arginine in position 170 would be indicated by
Arg170{Gly,Ala,Ser,Thr} or R170{G,A,S,T}
to indicate the variants
R170G, R170A, R170S, and R170T.
Deletions:
A deletion of glycine in position 195 is indicated by:
Gly195* or G195*
Similarly, the deletion of more than one amino acid residue, such as the deletion of glycine and leucine in positions 195 and 196 is designated
Gly195*+Leu196* or G195*+L196*
Insertions:
The insertion of an additional amino acid residue such as e.g. a lysine after G195 is designated:
Gly195GlyLys or G195GK; or
when more than one amino acid residue is inserted, such as e.g. a Lys, Ala and Ser after G195 this is shown as:
Gly195GlyLysAlaSer or G195GKAS (SEQ ID NO: 1)
In such cases the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example the sequences 194 to 196 would thus be:
In cases where an amino acid residue identical to the existing amino acid residue is inserted it is clear that a degeneracy in the nomenclature arises. If for example a glycine is inserted after the glycine in the above example this would be indicated by G195GG. The same actual change could just as well be indicated as A194AG for the change from
Such instances will be apparent to the skilled person. Thus, it is to be understood that the indication G195GG and corresponding indications encompass such equivalent degenerate indications.
Sometimes it is desired to both perform a modification and an insertion at the same position. This situation is also covered by the present definitions. Thus, S130TP indicates that the serine in position 130 has been replaced by a tyrosine and a proline. Another way to describe this variant is S130SP+S130T.
Filling a Gap:
Where a deletion in an enzyme exists in the reference comparison with the subtilisin BPNxe2x80x2 sequence used for the numbering, an insertion in such a position is indicated as:
*36Asp or *36D
for the insertion of an aspartic acid at position 36.
Multiple Modifications
Variants comprising multiple modifications are separated by pluses, e.g.:
Arg170Tyr+Gly195Glu or R170Y+G195E
representing modifications in positions 170 and 195 substituting tyrosine and glutamic acid for arginine and glycine, respectively, or e.g. Tyr167{Gly,Ala,Ser,Thr}+Arg170{Gly,Ala,Ser,Thr} designates the variants
This nomenclature is particularly relevant for designating modifications that are substitutions, insertions or deletions of amino acid residues having specific common properties, such as residues of positive charge (K, R, H), negative charge (D, E), or conservative amino acid modification(s) of e.g. Tyr167{Gly,Ala,Ser,Thr}+Arg170{Gly,Ala,Ser,Thr}, which signifies substituting a small amino acid for another small amino acid. See section xe2x80x9cDetailed description of the inventionxe2x80x9d for further details.
Numbering of Amino Acid Positions/Residues
For purposes of this invention, the numbering of amino acids corresponds to that of the amino acid sequence of subtilase BPNxe2x80x2 (BASBPN). For further description of the amino acid sequence of subtilisin BPNxe2x80x2, see FIGS. 1 and 2, or Siezen et al., Protein Engng., 4, 719-737 (1991).
Proteases
Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W. H. Freeman and Company, San Francisco, Chapter 3).
Serine Proteases
A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, xe2x80x9cPrinciples of Biochemistry,xe2x80x9d Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272 (1973)).
The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Dalton range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest, Bacteriological Rev., 41, 711-753 (1977)).
Subtilases
A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al., Protein Engng., 4, 719-737 (1991) and Siezen et al., Protein Science, 6, 501-523 (1997). They are defined by homology analysis of more than 170 amino acid sequences is of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously often defined as a serine protease produced by gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases has been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al. (1997).
One subgroup of the subtilases, I-S1 or xe2x80x9ctrue#xe2x80x9d subtilisins, comprises the xe2x80x9cclassicalxe2x80x9d subtilisins, such as subtilisin 168 (BSS168), subtilisin BPNxe2x80x2, subtilisin Carlsberg (ALCALASE(copyright), NOVO NORDISK A/S), and subtilisin DY (BSSDY).
A further subgroup of the subtilases, I-S2 or high alkaline subtilisins, is recognized by Siezen et al. Sub-group I-S2 proteases are described as highly alkaline subtilisins and comprises enzymes such as subtilisin PB92 (BAALKP) (MAXACAL(copyright), Gist-Brocades NV), subtilisin 309 (SAVINASE(copyright), NOVO NORDISK A/S), subtilisin 147 (BLS147) (ESPERASE(copyright), NOVO NORDISK A/S), and alkaline elastase YaB (BSEYAB).
List of Acronyms for Subtilases:
I-S1
Subtilisin 168, BSS168 (BSSAS (Subtilisin amylosacchariticus)), BSAPRJ (Subtilisin J), BSAPRN (Subtilisin NAT), BMSAMP (Mesentericopeptidase),
Subtilisin BPNxe2x80x2, BASBPN,
Subtilisin DY, BSSDY,
Subtilisin Carlsberg, BLSCAR (BLKERA (Keratinase), BLSCA1, BLSCA2, BLSCA3),
BSSPRC, Serine protease C
BSSPRD, Serine protease D
I-S2
Subtilisin Sendai, BSAPRS
Subtilisin ALP 1, BSAPRQ,
Subtilisin 147, Esperase(copyright), BLS147 (BSAPRM (SubtilisinAprM), BAH101)
Subtilisin 309, SAVINASE(copyright), BLS309/BLSAVI (BSKSMK (M-protease), BAALKP (Subtilisin PB92, Bacillus alkalophilic alkaline protease), BLSUBL (Subtilisin BL)),
Alkaline elastase YaB, BYSYAB
xe2x80x9cSAVINASE(copyright)xe2x80x9d
SAVINASE(copyright) is marketed by NOVO NORDISK A/S. It is subtilisin 309 from B. lentus and differs from BAALKP only in one position (N87S, see FIG. 1). SAVINASE(copyright) has the amino acid sequence designated b) in FIG. 1.
Parent Subtilase
The term xe2x80x9cparent subtilasexe2x80x9d describes a subtilase defined according to Siezen et al. (1991 and 1997). For further details see description of xe2x80x9cSUBTILASESxe2x80x9d immediately above. A parent subtilase may also be a subtilase isolated from a natural source, wherein subsequent modification have been made while retaining the characteristic of a subtilase. Alternatively the term xe2x80x9cparent subtilasexe2x80x9d may be termed xe2x80x9cwild-type subtilasexe2x80x9d.
Modification(s) of a Subtilase Variant
The term xe2x80x9cmodification(s)xe2x80x9d used herein is defined to include chemical modification of a subtilase as well as genetic manipulation of the DNA encoding a subtilase. The modification(s) can be replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertions in or at the amino acid(s) of interest.
Subtilase Variant
In the context of this invention, the term subtilase variant or mutated subtilase means a subtilase that has been produced by an organism which is expressing a mutant gene derived from a parent microorganism which possessed an original or parent gene and which produced a corresponding parent enzyme, the parent gene having been mutated in order to produce the mutant gene from which said mutated subtilase protease is produced when expressed in a suitable host.
Homologous Subtilase Sequences
The present invention relates to modified subtiliases comprising an insertion in the active site loop (b) region in the subtilase SAVINASE and other parent (wild-type) subtilases, which have a homologous primary structure to that of SAVINASE(copyright). The homology between two amino acid sequences is in this context described by the parameter xe2x80x9cidentityxe2x80x9d.
In order to determine the degree of identity between two subtilases the GAP routine of the GCG package version 9.1 can be applied using the same settings as indicated above. The output from the routine is besides the amino acid alignment the calculation of the xe2x80x9cPercent Identityxe2x80x9d between the two sequences.
Based on this description it is routine for a person skilled in the art to identify suitable homologous subtilases and corresponding homologous active site loop regions, which can be modified according to the invention.
Wash Performance
The ability of an enzyme to catalyze the degradation of various naturally occurring substrates present on the objects to be cleaned during e.g. wash or hard surface cleaning is often referred to as its washing ability, wash-ability, detergency, or wash performance. Throughout this application the term wash performance will be used to encompass this property.
Isolated DNA Sequence
The term xe2x80x9cisolatedxe2x80x9d, when applied to a DNA sequence molecule, denotes that the DNA sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5xe2x80x2 and 3xe2x80x2 untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature, 316, 774-78 (1985)). The term xe2x80x9can isolated DNA sequencexe2x80x9d may alternatively be termed xe2x80x9ca cloned DNA sequencexe2x80x9d.
Isolated Protein
When applied to a protein, the term xe2x80x9cisolatedxe2x80x9d indicates that the protein has been removed from its native environment.
In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e. xe2x80x9chomologous impuritiesxe2x80x9d (see below)).
An isolated protein is greater than 10% pure, preferably greater than 20% pure, more preferably greater than 30% pure, as determined by SDS-PAGE. Further it is preferred to provide the protein in a highly purified form, i.e., greater than 40% pure, greater than 60% pure, greater than 80% pure, more preferably greater than 95% pure, and even more preferably greater than 99% pure, as determined by SDS-PAGE.
The term xe2x80x9cisolated proteinxe2x80x9d may alternatively be termed xe2x80x9cpurified proteinxe2x80x9d.
Homologous Impurities
The term xe2x80x9chomologous impuritiesxe2x80x9d means any impurity (e.g. a polypeptide other than the polypeptide of the invention) which originates from the homologous cell where the polypeptide of the invention is originally obtained from.
Obtained from
The term xe2x80x9cobtained fromxe2x80x9d as used herein in connection with a specific microbial source, means that the polynucleotide and/or polypeptide is produced by the specific source, or by a cell in which a gene from the source has been inserted.
Substrate
The term xe2x80x9cSubstratexe2x80x9d used in connection with a substrate for a protease should be interpreted in its broadest form as comprising a compound containing at least one peptide bond susceptible to hydrolysis by a subtilisin protease.
Product
The term xe2x80x9cproductxe2x80x9d used in connection with a product derived from a protease enzymatic reaction should in the context of this invention be interpreted to include the products of a hydrolysis reaction involving a subtilase protease. A product may be the substrate in a subsequent hydrolysis reaction.